Dietary fat modification is a promising approach to prevent age-related macular degeneration (AMD). However, which types of fatty acids carry a greater risk for AMD remains unclear. In this study, we compared the effects of 18-carbon fatty acids with different degrees of unsaturation on the expression of the proinflammatory genes cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in human retinal pigment epithelium (RPE). Additionally, we investigated whether lutein could modulate these genes induced by fatty acids in RPE. Treatment with oleic acid, linoleic acid (LA), or linolenic acid increased the expression of iNOS and COX-2 genes and the production of prostaglandin E2 and nitric oxide (NO) in RPE, whereas the saturated fatty acid stearic acid had little effect on these genes. Of the fatty acids studied, LA had the greatest effects on the induction of these genes. Furthermore, LA also induced NF-κB transcriptional activation the most. Lutein inhibited LA-induced expression of COX-2 and iNOS in a dose-dependent manner. These data suggested that specific unsaturated fatty acids, particularly LA, can stimulate RPE cells to express proinflammatory genes, which may contribute to the pathogenesis of AMD. Lutein inhibited the expression of these genes induced by LA through blockade of NF-κB activation.